Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a lar...Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a large genome,contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein.Furthermore,animal alphaherpesviruses have a wide host spectrum,can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen,thus making them ideal carriers for recombinant virus-vectored vaccines.With the development of gene-editing technology,recombinant viruses capable of expressing foreign genes can be constructed by various methods.Currently,studies on recombinant virusvectored vaccines constructed based on animal alphaherpesviruses have involved poultry,pigs,cattle,sheep,and companion animals.Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases.This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines,aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.展开更多
AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bact...AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.展开更多
AIM:To identify and understand the regular distribution pattern for Salmonella enteritidis (S. enteritidis) in the internal organs of mice after an oral challenge over a 3 wk period. METHODS:Assays based on the serova...AIM:To identify and understand the regular distribution pattern for Salmonella enteritidis (S. enteritidis) in the internal organs of mice after an oral challenge over a 3 wk period. METHODS:Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS:The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms. CONCLUSION:The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteritidis infection in vivo.展开更多
AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied...AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied to analyze the intestinal microbial community diversity and dynamic change including duodenum, jejunum, ileum, cecum and rectum from healthy ducklings and 7-day-old ducklings after oral infection with S. enteritidis at different time points.RESULTS: The intestinal microbial community of the control healthy ducklings was steady and the ERIC-PCR band numbers of the control healthy ducklings were the least with rectum and were the most with caecum. ERIC-PCR bands of orally inoculated ducklings did not obviously change until 24 h after inoculation (p.i.). The numbers of the ERIC-PCR bands gradually decreased from 24 h to 72 h p.i., and then, with the development of disease, the band numbers gradually increased until 6 d p.i. The prominent bacteria changed because of S. enteritidis infection and the DNAstar of staple of ERIC-PCR showed that aerobe and facultative aerobe (Escherichia coli, Shigella, Salmonella) became preponderant bacilli in the intestine of orally infected ducklings with SE.CONCLUSION: This study has provided significant data to clarify the intestinal microbial community diversity and dynamic change of healthy and S. enteritidis orally infected ducklings, and valuable insight into the pathogenesis of S. enteritidis infection in both human and animals.展开更多
AIM:To detect Salmonella enteritidis (S. enteritidis) in paraffin slices and antigen location in infected duck tissues. METHODS:Rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody,...AIM:To detect Salmonella enteritidis (S. enteritidis) in paraffin slices and antigen location in infected duck tissues. METHODS:Rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method (IFA) was established to detect the S. enteritidis antigen in paraffin slices. S. enteritidis was detected in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS:The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen was mainly distributed in the infected cell cytoplasm.CONCLUSION:IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.展开更多
Many mammalian herpes viruses utilize heparan sulfate(HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C(gC) homologues. However, our un...Many mammalian herpes viruses utilize heparan sulfate(HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C(gC) homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus(DPV) remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus(DPV-ΔgC-EGFP). Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins(g B-DPV, gC-DPV, and g E-DPV) to HS was assessed using a prokaryotic expression system and HiTrap^(TM) Heparin HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-ΔgC-EGFP or wild-type strain Chinese virulent duck plague virus(DPV-CHv). The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.展开更多
New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for univ...New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.展开更多
Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and cap...Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.展开更多
CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains...CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for specific detection of goose CD8α(go CD8α). The results showed that the optimal coated antibody and antigen dilutions were 1:50(the antibody titer was 1:12 800) and 1:32(0.3 ng m L^(–1)), respectively, while the optimal capture antibody and horseradish peroxidase(HRP)-labelled goat anti-rabbit Ig G dilutions were 1:50(the antibody titer was 1:51 200) and 1:4 000(the antibody titer was 1:5 000), respectively. The optimal blocking buffer was 5% bovine serum albumin(BSA). The best incubating condition was overnight at 4°C, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^(–3) ng m L^(–1). Most importantly, go CD8α expression levels in goose spleen mononuclear cells(MNCs) post-Goose parvoviruse(GPV) infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.展开更多
基金supported by grants from the National Key Research and Development Program of China(2017YFD0500800)the earmarked fund for China Agriculture Research System(CARS-42-17)+1 种基金the Integration and Demonstration of Key Technologies for Goose Industrial Chain in Sichuan Province,China(2018NZ0005)the Sichuan Veterinary Medicine and Drug Innovation Group of China Agriculture Research System(SCCXTD-2020-18)。
文摘Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a large genome,contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein.Furthermore,animal alphaherpesviruses have a wide host spectrum,can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen,thus making them ideal carriers for recombinant virus-vectored vaccines.With the development of gene-editing technology,recombinant viruses capable of expressing foreign genes can be constructed by various methods.Currently,studies on recombinant virusvectored vaccines constructed based on animal alphaherpesviruses have involved poultry,pigs,cattle,sheep,and companion animals.Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases.This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines,aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.
基金The National Science Technology Pillar Program, 2007Z06-017Program for New Century Outstanding Talents from Universities, NCET-04-0906/NCET-06-0818+1 种基金Sichuan Provincial Basic Research Program, 04JY029-006-1/04JY021-100/07JY029-017Program for Key Disciplines Construction of Sichuan Province, SZD0418
文摘AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.
基金The National Key Technology R&D Program of China, No. 2004BA901A03National Scientific and Technical Support Program, No. 2007Z06-017+3 种基金The Cultivation Fund of the Key Scientific and Technical Innovation Project & Ministry of Education of China, No. 706050Program for New Century Excellent Talents in University, No. NCET-04-0906/NCET-06-0818Sichuan Province Basic Research Program, No. 04JY0290061/07JY029-017Program for Key DisciplinesConstruction of Sichuan Province No. SZD0418
文摘AIM:To identify and understand the regular distribution pattern for Salmonella enteritidis (S. enteritidis) in the internal organs of mice after an oral challenge over a 3 wk period. METHODS:Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS:The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms. CONCLUSION:The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteritidis infection in vivo.
基金The National Science &Technology Pillar Program, 2007Z06-017Program for New Century Excellent Talents in University, NCET-04-0906/NCET-06-0818+1 种基金Sichuan Province Basic Research Program, 04JY029-006-1/04JY021-100/07JY029-017Program for Key Disciplines Construction of Sichuan Province, SZD0418
文摘AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied to analyze the intestinal microbial community diversity and dynamic change including duodenum, jejunum, ileum, cecum and rectum from healthy ducklings and 7-day-old ducklings after oral infection with S. enteritidis at different time points.RESULTS: The intestinal microbial community of the control healthy ducklings was steady and the ERIC-PCR band numbers of the control healthy ducklings were the least with rectum and were the most with caecum. ERIC-PCR bands of orally inoculated ducklings did not obviously change until 24 h after inoculation (p.i.). The numbers of the ERIC-PCR bands gradually decreased from 24 h to 72 h p.i., and then, with the development of disease, the band numbers gradually increased until 6 d p.i. The prominent bacteria changed because of S. enteritidis infection and the DNAstar of staple of ERIC-PCR showed that aerobe and facultative aerobe (Escherichia coli, Shigella, Salmonella) became preponderant bacilli in the intestine of orally infected ducklings with SE.CONCLUSION: This study has provided significant data to clarify the intestinal microbial community diversity and dynamic change of healthy and S. enteritidis orally infected ducklings, and valuable insight into the pathogenesis of S. enteritidis infection in both human and animals.
基金the National Key Technology R&D Program of China, No. 2004BA 901A 03National Scientific and Sechnical Support Program, No. 2007Z06-017+3 种基金The Cultvation Fund of the Key Scientific and Technical Innovation Project & Ministry of Education of China, No. 706050Program for New Century Excellent Talents in University, No. NCET-04-0906/NCET-06-0818Sichuan Province Basic Research Program, No. 04JY0290061/07JY029-017Program for Key Disciplines Construction of Sichuan Province No. SZD0418
文摘AIM:To detect Salmonella enteritidis (S. enteritidis) in paraffin slices and antigen location in infected duck tissues. METHODS:Rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method (IFA) was established to detect the S. enteritidis antigen in paraffin slices. S. enteritidis was detected in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS:The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen was mainly distributed in the infected cell cytoplasm.CONCLUSION:IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.
基金supported by the grants from the National Natural Science Foundation of China(31072157)the National Key Technologies R&D Program of China during the 12th Five-Year Plan period(2015BAD12B05)+1 种基金the Foundation of China Agricultural Research System(CARS-43-8)the Major Project of Education Department in Sichuan,China(16ZA0027)
文摘Many mammalian herpes viruses utilize heparan sulfate(HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C(gC) homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus(DPV) remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus(DPV-ΔgC-EGFP). Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins(g B-DPV, gC-DPV, and g E-DPV) to HS was assessed using a prokaryotic expression system and HiTrap^(TM) Heparin HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-ΔgC-EGFP or wild-type strain Chinese virulent duck plague virus(DPV-CHv). The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.
文摘New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.
基金supported by the National Key Technology R&D Program of China(2015BAD12B05)the earmarked fund for China Agricultural Research System(CARS-43-8)+1 种基金the Integration and Demonstration of Key Technologies for Duck Industry in Sichuan Province,China(2014NZ0030)the Sichuan Province Research Programs,China(2014-002)
文摘Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.
基金funded by the National Natural Science Foundation of China (31201891)the Ph D Programs Foundation of Ministry of Education of China (20125103120012)+3 种基金the Innovative Research Team Program in Education Department of Sichuan Province, China (2013TD0015)the National Key Technology R&D Program of China (2015BAD12B05)the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province, China (2014NZ0030)the China Agricultural Research System (CARS-43-8)
文摘CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for specific detection of goose CD8α(go CD8α). The results showed that the optimal coated antibody and antigen dilutions were 1:50(the antibody titer was 1:12 800) and 1:32(0.3 ng m L^(–1)), respectively, while the optimal capture antibody and horseradish peroxidase(HRP)-labelled goat anti-rabbit Ig G dilutions were 1:50(the antibody titer was 1:51 200) and 1:4 000(the antibody titer was 1:5 000), respectively. The optimal blocking buffer was 5% bovine serum albumin(BSA). The best incubating condition was overnight at 4°C, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^(–3) ng m L^(–1). Most importantly, go CD8α expression levels in goose spleen mononuclear cells(MNCs) post-Goose parvoviruse(GPV) infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.
